Decrease in ATP availability in platelets takes place during metabolic blockage or storage and is associated with loss of functional response of the cells. This suggests that ATP consuming processes are involved in execution of platelet function and/or maintenance of resting cells in a responsive state. We intend to study the function-related behavior of actin and myosin in resting and stimulated, intact platelets, using these approaches: Actin: Considerable amounts of metabolic ADP presumably are bound to actin, and the compond is ethanol insoluble. Isotope experiments show that ADP in the complex is turning over rapidly in the intact platelet. We believe this represents an ATP consuming process, involving only actin, that keeps G- and F-actin in a certain proportion which is optimal for the sudden interaction of actin with myosin upon stimulation of the platelets. The turnover of the ADP-actin complex would therefore be determining for platelet responsiveness. We will establish the identity of this protein and study the kinetics of its labeling with radioactive ADP in different functional states in intact platelets and in the material purified from platelets. Myosin: Platelet myosin light chain (MLC, 20,000 daltons) can be phosphorylated by ATP a protein kinase. Such phosphorylated myosin has a markedly increased actin-stimulated ATPase, which presumably is the ATPase operating during contraction. The MLC is phosphorylated in intact platelets in response to thrombin and prior to secretion, suggesting activation of myosin ATPase prior to execution of a function. We will correlate the degree of MLC phosphorylation in intact platelets with the degree of functional response to the usual inducers of platelet function. The kinetics and dose-response curves will be determined for these inducers and the regulation of MLC phosphorylation will be studied. These studies will elucidate the molecular mechanisms of the ATP-utilizing processes that are involved in platelet function.